A2A adenosine receptor (AR) activation inhibits pro-inflammatory cytokine production by human CD4+ helper T cells and regulates Helicobacter-induced gastritis and bacterial persistence

Abstract

Helicobacter pylori causes a lifelong infection and provides a model of bacterial adaptation and persistent colonization. Adenosine is an anti-inflammatory mediator that limits tissue damage during inflammation. We studied the role of adenosine in the T-cell-mediated regulation of gastritis and bacterial persistence. After 4 h of activation, human T helper (Th) cells increased A(2A) adenosine receptor (A(2A)AR) mRNA level (sevenfold). A(2A)AR was the predominant subtype expressed in resting and stimulated gastric or peripheral Th cells. Stimulation with ATL313, an A(2A)AR agonist, increased cyclic AMP (cAMP) accumulation and reduced interleukin-2 (IL-2) production by 20-50%. ATL313 also attenuated tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) production, which was inhibited by an A(2A)AR antagonist. Infection of IL-10-deficient mice with H. pylori is cleared spontaneously due to the marked inflammation. Administration of ATL313 during infection reduced gastritis and pro-inflammatory cytokine responses while bacterial load increased. In contrast, infection of A(2A)AR-deficient mice enhanced gastritis. Thus, A(2A)AR limits the pro-inflammatory effects of Th cells and favor chronic Helicobacter infection.

Figure 1

A_2AAR is the predominant isoform expressed in T cells. AR expression was assessed in (a) Jurkat cells and reported as the fold change in mRNA in stimulated T cells relative to unstimulated T cells at the indicated time points. Subsequently, the expression of AR in (b) purified human CD4⁺ Th cells or preparations of (c) gastric lamina propria lymphocytes (LPL) was assessed and reported as the increase in mRNA in stimulated T cells relative to unstimulated T cells at the resting time point. In each case, T cells were activated with PMA + inomycin (50 + 500 ng ml⁻¹, respectively) at 37 ° C for varying lengths of time, and A₁AR, A_2AAR, A_2BAR and A₃AR mRNA expressions were measured by real-time RT-PCR. Data shown are the mean ± s.e.m. from three independent experiments. * Significant difference (P < 0.05) between unstimulated and stimulated cells.

Figure 2

A selective A_2AAR agonist, ATL313, triggers cAMP accumulation in helper T cells. Purified human CD4⁺ Th cells were activated for 2h and treated for 10 min with 1 U ml ⁻¹ ADA in the presence of vehicle or 1 µM rolipram±1,000 nm ATL313 or forskolin, a positive control for cAMP accumulation. Intracellular cAMP accumulation was measured using a chemiluminescent immunoassay system. Data shown are the mean±s.e.m. from three independent experiments performed in triplicate. * Significant difference (P < 0.05) between the ATL313-treated and ATL313-untreated cells.

Figure 3

Suppression of IL-2 expression by CD4⁺ Th cells is mediated through the A_2A adenosine receptor. Purified human CD4⁺ T cells were activated in plates coated with 5 µg ml⁻¹ anti-CD3/CD28 mAb with 1U ml ⁻¹ ADA or after the addition of the nonselective A_2AAR agonist, (a) NECA, or a selective A_2AAR agonist, (b) ATL313, and IL-2 protein was measured by ELISA in supernatants collected after 24h and expressed as the production relative to stimulated Th cells in the absence of either NECA or ATL313. Data shown are the mean ± s.e.m. from three or four independent experiments performed in triplicate. * Significant difference (P < 0.05) between the NECA- or ATL313-treated and -untreated cells.

Figure 4

Inhibition of IL-2 production by ATL313. (a) Purified human CD4⁺ T cells were incubated on immobilized anti-CD3/CD28 mAb with 1 U ml⁻¹ ADA (Stimulation Control) or with adenosine analogs (ATL313, NECA) in the presence or absence of A_2A antagonists (ZM241385 or SCH58261 = SCH) or ATL801, an A_2B antagonist. Rolipram, a phosphodiesterase inhibitor, was used to allow cAMP to accumulate. Supernatants were collected after 24 h, and IL-2 concentrations were determined by ELISA and expressed as described in Figure 3. ELISA data shown are the mean±s.e.m. from three or four independent experiments performed in triplicate. (b) CD4⁺ T cells were cultured as described in panel a, but in this case, another A_2A agonist, ATL146e, was also used. Cells were collected after 24h before IL-2 mRNA was measured by quantitative RT-PCR. Activated control refers to CD4⁺ T lymphocytes activated in the absence of any drugs. mRNA data shown are from a single experiment performed in triplicate, representative of three independent experiments. * Significant difference (P < 0.05) of the treatment groups compared with the “ stimulation control. ”

Figure 5

Suppression of pro-inflammatory cytokines by A_2AAR agonists. CD4⁺ T cells were stimulated and supernatants were collected after 24 h. Subsequently, (a) IFN-γ, (b) TNF-α, and (c) IL-4 were measured using a mutiplex bead array. The effects of the A_2AAR agonist (ATL313) were also compared in the presence of an A_2AAR antagonist (ZM241385). Data shown are from a single experiment performed in triplicate, representative of three independent experiments. ND = not detectable. * Significant difference (P < 0.05) of the treatment groups compared with the “ stimulation control.

Figure 6

Suppression of pro-inflammatory cytokine production by gastric T cells is partially mediated through the A_2A adenosine receptor. LPL isolated from human gastric biopsy specimens were incubated on immobilized anti-CD3/CD28 mAb with 1 U ml⁻¹ ADA or in the presence of the A_2AAR agonist, ATL313. Supernatants were collected after 24h, and (a-c) IL-2, IFN-γ, and TNF-α proteins were measured using a mutiplex bead array and expressed as the production relative to stimulated Th cells in the absence of ATL313. Cytokine protein data shown are the mean±s.e.m. from three independent experiments performed in triplicate. IL-2 mRNA was extracted from T cells (from panels a-c) and assayed by real-time RT-PCR (d). mRNA data shown are from a single experiment performed in triplicate, representative of three independent experiments. * Significant difference (P <0.05) between the ATL313- treated and -untreated cells.

Figure 7

Gastritis is more severe in Helicobacter felis-infected A_2AAR-deficient mice. Mice were infected by gavage with 1 × 10⁸ CFU of H.felis per inoculation every other day for three separate inoculations and euthanized 4 weeks later. Gastric tissue was processed for histology and H. felis colonization was assessed. (a) Myeloperoxidase (MPO) (left panel) and H & Estained (two right panels with inset from center panel shown at higher magnification in the outer right panel) of gastric sections from representative uninfected or infected C57BL/6 (upper panels), and uninfected or infected A_2AAR −/− (lower panels) mice. Arrows indicate MPO-expressing cells or MNC (original magnification of × 100). Only a few scattered MNC-and MPO-positive granulocytes can be seen in the submucosa and lamina propria with no abnormal thickening of the gastric wall of uninfected control mice. Severe gastritis with dense MNC infiltration and diffuse MPO-positive granulocytes in the submucosa and mucosa of the infected A_2AAR^−/− mice in both the cardia and antral regions. MNCs aggregate between the glands are spanning the entire width of the mucosa in some cases. A concomitant thickening of the gastric wall as well as gastric of glandular atrophy can be observed. (b) Overall gastritis score in uninfected and infected wild-type and A_2AAR^−/− mice. (c) Regional gastritis scores in infected wild-type and A_2AAR^−/− mice. (d) Quantitative expression of MPO-positive cells in uninfected and infected, control, and A_2AAR^−/− mice expressed per ×200 field. (e) H. felis colonization in gastric tissue analyzed by the presence of H. felis-specific UreA gene. Data are mean±s.e.m. from pooled data including two identical experiments with four mice per group. * Significant difference (P < 0.05) between the wild-type and A_2AAR^−/− mice.

Figure 8

Oral administration of an A_2AAR agonist in Helicobacter pylori-infected IL-10^−/− mice reduces gastric inflammation but increases bacterial load. IL-10-deficient mice were infected as described in Figure 7. All mice were given either vehicle control chow (ATL313(−), n = 4) or ATL313 chow (ATL313(+), n = 4) for 1 week before infection and continued until killing. Mice were killed 7(7d.p.i.) or 15 days (15d.p.i.) later, and gastric tissue (each longitudinal section) was processed for histology, MPO staining, mucosal cytokine expression, and H. pylori colonization by colony count. (a) Myeloperoxidase (MPO) (left panel) and H&E stained (two right panels with inset from center panel shown at higher magnification in the outer right panel) of gastric sections from representative uninfected IL-10^−/− (upper panels), and infected IL-10^−/− without (middle panels) or with ATL313 chow-treated mice (lower panels). Arrows indicate MPO-expressing cells (original magnification of × 100). Some scattered mononuclear cells (MNCs) and MPO-positive granulocytes can be seen in the submucosa and lamina propria, with no abnormal thickening of the gastric wall of uninfected control IL-10-deficient mice. Severe gastritis with dense MNC infiltration and diffuse MPO-positive granulocytes in the submucosa and mucosa of the untreated infected mice in both cardia and antral regions. A concomitant hyperplasia with widespread thickening of the gastric wall as well as of gastric atrophy can be observed. ATL313-treated mice are almost normal with few inflammatory cells infiltration. (b) Histological scoring of gastritis, (c) quantitative expression of MPO-positive cells expressed per field, and (d) mucosal TNF-α, and (e), IFN-γ production in gastric tissue of in H. pylori-infected IL-10-deficient mice without or with ATL313 treatment. (f) H. pylori colonization in gastric tissue measured by colony count on days 7 and 15 after infection with same treatment as mentioned earlier. Data are mean±s.e.m. from one experiment with four mice per group that was carried out at least twice with similar results. * Significant difference (P < 0.05) between the untreated and treated mice.